The present invention relates to synthetic reagents or substrates which are used for the quantitative determination of proteolytic enzymes. More particularly, the invention relates to sarcosylproplylarginine-p-nitroanilide derivatives which are useful as reagents for the quantitative determination of proteolytic enzyme which split peptide chains on the carboxyl side of arginine in human and mammal body fluids as well as in vegetable and animal cell extracts and in glandular venoms of cold-blooded animals such as snakes.
U.S. Pat. No. 4,070,245 describes glycylprolylarginine-p-nitroanilide derivatives useful in chromogenic substrates for determining proteolytic enzymes in biological fluids. Compounds of the present invention differ from the prior art in that they terminate with a sarcosine or sarcosine derivative while prior art compounds terminate with glycine or glycine derivatives.
The enzymatic hydrolysis reaction can be represented by the following scheme: ##STR2## E=enzyme S=substrate
ES=enzyme-substrate complex PA1 P.sub.1 and P.sub.2 =products PA1 k.sub.1 k.sub.2 k.sub.cat and k.sub.4 =rate constants
Dissociation constant for EQU ES=k.sub.2 /k.sub.1 =K.sub.m (Michaelis constant)
If [S]&gt;&gt;[E] and k.sub.4&lt;&lt;k.sub.cat, the following is true: ##EQU1## The rate constant at which chromophore P.sub.1 is formed is: v=k.sub.cat.multidot.[ES] ##EQU2## If E is completely bound to S, then [ES]=[E] and ##EQU3## Lineweaver-Burk equation: ##EQU4##
As is evident from equation (2) constants K.sub.m and k.sub.cat determine the activity of the enzyme substrate for a given enzyme. For determining these constants, the following procedure is followed.
The enzyme and the substrate are mixed in a buffer solution, and the reaction is followed spectrophotometrically for 2 to 30 minutes. The concentration of substrate [S] is varied, whereas the enzyme concentration [E] is kept constant. If the extinction (OD) (=optical density) is plotted in a coordinate system as a function of time, a curve is obtained the tangent of which (difference in extinction per minute, .DELTA. OD/minute, from which the quantity in .mu.moles of pNA/min(v) can be calculated) at time zero corresponds to the initial course of the hydrolysis. By means of this tangent the initial rate of the hydrolysis can be determined.
If l/v is plotted against l/[S], a Lineweaver-Burk diagram is obtained from which v.sub.max and K.sub.m can be determined graphically. Thus K.sub.m and k.sub.cat can be determined.